Leptotrichia buccalis ATCC 14201 is a Gram-negative, anaerobic rod-shaped bacterium resident in oral biofilm at the tooth surface. The sequenced genome of this organism reveals three contiguous genes at loci: Lebu_1525,1526 and 1527. The translation products of these genes exhibit significant homology with phospho-&#945;-glucosidase (Pagl), a regulatory protein (GntR) and a phosphoenol pyruvate-dependent sugar transport protein (EIICB), respectively. In non-oral bacterial species, these genes comprise the sim operon that facilitates sucrose isomer metabolism. Growth studies showed that L. buccalis fermented a wide variety of carbohydrates, including four of the five isomers of sucrose. Growth on the isomeric disaccharides elicited expression of a 50kDa polypeptide comparable in size to that encoded by Lebu_1525. The latter gene was cloned, and the expressed protein was purified to homogeneity from Escherichia coli TOP 10 cells. In the presence of two cofactors, NAD+ and Mn2+ ion, the enzyme readily hydrolyzed p-nitrophenyl-&#945;-glucopyranoside 6-phosphate (pNP&#945;G6P), a chromogenic analog of the phosphorylated isomers of sucrose. By comparative sequence alignment, immuno-reactivity and signature motifs, the enzyme can be assigned to the phospho-&#945;-glucosidase (Pagl) clade of Family 4 of the glycosyl hydrolase super family. We suggest that the products of Lebu_1527 and 1525, catalyze the phosphorylative translocation and hydrolysis of sucrose isomers in L. buccalis, respectively. Four genetically diverse, but 16S rDNA related species of Leptotrichia have recently been described: L. goodfellowii, L. hofstadii, L. shahii and L. wadei. The phenotypic traits of these new species, with respect to carbohydrate utilization, have also been determined.The results of our investigation have been submitted, and accepted, for publication in the journal MOLECULAR ORAL MICROBIOLOGY.